Lior schenk - pkar subunits exhibit differential localizations and functions in the brain
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Lior schenk - pkar subunits exhibit differential localizations and functions in the brain
- 1. PKAR Subunits Exhibit Differential Localizations and Functions in the Brain Lior Schenk R. Ilouz, S. Taylor, T. Stiles, L. Schenk, J.L. Goldberg UC San Diego Division of Biological Sciences
- 2. RCSB Protein Data Bank (http://www.rcsb.org/pdb/101/motm.do?momID=152) C C R R Inactive PKA R- Regulatory subunit C- Catalytic Subunit C C R R cAMP cAMP and the PKA holoenzyme How does the differential expression of PKAR isoforms affect LTP induction?
- 3. PKA Signaling System PKA: cAMP dependent protein kinase Hormone GPCR Adenylate Cyclase ATP C C C C R R R R Inactive PKA DNA CRE CREB R- Regulatory subunit C- Catalytic Subunit βγ α γ β α cAMP Transcription Cytosol R R Functions: Memory NT Metabolism Glycogen Metabolism Steroid Metabolism Muscle Contraction R. Ilouz
- 4. Variable expression of R1b and R2b throughout the brain Images courtesy R. Ilouz R1b k/o: LTP and LTD impairment R2b k/o: “lean” phenotype Noticeably different localization in CA3 vs CA1 cells
- 5. Connecting the dots… • cAMPPKA pCREBL-LTP • Which PKA isoforms are responsible? • Hypothesis: R1b and R2b, which differentially localize in the hippocampus, play dissimilar roles in the induction of LTP.Kandel; Squire (2000). Memory: From Mind to Molecules. Does loss of R1b or R2b function lead to a loss of pCREB, which is necessary for L-LTP?
- 6. Experimental Procedure • Hippocampal neuron prep (courtesy R Ilouz) • GFP-shRNA knockdowns of R1/R2 subunits • Forskolin treatment elevated cAMP PKA pCREB cL-LTP • Immunofluorescence – Transfection backbone – α-CREB / α-pCREB – DAPI Irr R1b R2b CREB p-CREB
- 7. RESULTS
- 8. irr: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI pCREB levels are visibly decreased in R1b fields.
- 9. irr: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI pCREB levels are visibly decreased in R1b fields.
- 10. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI 1, 2, 3, 5, 6, 7, 9, 10 pCREB levels are visibly decreased in R1b fields.
- 11. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI 1, 2, 3, 5, 6, 7, 9, 10 pCREB levels are visibly decreased in R1b fields.
- 12. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI 1, 2, 3, 5, 6, 7, 9, 10 pCREB levels are visibly decreased in R1b fields.
- 13. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI pCREB levels are visibly decreased in R1b fields. 1, 2, 3, 5, 6, 7, 9, 10
- 14. Data quantification Fluorescence Quantification (in ImageJ) • Select nuclear area (freeform selection tool, DAPI channel) • Transpose selection onto pCREB channel • Analyze Mean Gray Value
- 15. 0 50 100 150 200 250 irr r1b r2b pCREB mean fluorescence
- 16. 0 50 100 150 200 250 irr r1b r2b pCREB mean fluorescence
- 17. DISCUSSION pCREB levels are visibly decreased in R1b fields. Specific interference of R1b may lead to cL-LTP impairment. cAMP PKA pCREB L-LTP 0 50 100 150 200 250 irr r1b r2b pCREB mean fluorescence irr R1b R2b
- 18. Future Considerations • Follow-up to reproduce findings – Implement optimized vector more transfected cells, more useful control • Introduce new stain – B3-tubulin @ cy5 – Identify add’l neurons + transfection efficiency • Increase cell density – More synapses, branching – Cell-to-cell comparison
- 19. Thank you!